Development Of Molecular ELISA Method For Diagnosing Anaplasimasis Caused By Anaplasma Marginale And PCR Method For Diagnosing Eperythrozoon Wenyonii

Anaplasmosis and Eperythrozoonosis are the two kinds of main parasite disease. Theclinical sympotoms of the two disease are anaemia, jaundice, anepithymia, athrepsy andso on. Anaplasma marginale and eperythrozoon are the parasite which parasitize in theblood of the animal. It is very difficult to classify the two parasites by microscopy. So wesought to develop laboratory method to distinguish them.Anaplasmosis is a hemoparasite and tick-borne disease of cattle, sheep and otherruminants caused by Anaplasma marginale. Anaplasmosis can be found all over theworld and mainly cause the death of cattle, sheep, deer and other ruminants. The mortalityof cattle can be over 80%. It is one of the most important tick-borne diseases in theanimal quarantine. In this study,the partial MSP5 gene of Anaplasma marginale wascloned and expressed by prokaryotic vector in E. coli. Then the target protein was purifiedand used to develop ELISA. The ELISA method was used to study the epidemiology ofanaplasmosis in Hubei province.Using a pair of specific primers designed according to the relevant nucleotidesequence (AY714547) from GenBank, the MSP5 gene of Anaplasma marginale wasamplified with PCR method. The PCR product was cloned into pGEX-KG to construct aprokaryotic recombinant plasmid KG-MSP5. Then the recombinant plasmid KG-MSP5was transformed into Escherichia coli BL21. The target 46kD protein was found in theIPTG-inducible product and finally purified by BugBuster GST Bind Purification Kit.Western blot analysis proved the recombinant protein had good reactive ability against A.marginale positive serum.An indirect ELISA was developed using recombinant MSP5 protein as describedpreviously, which had high specificity to A. marginale. The 478 blood samples wereexamined synchronously by iELISA and cELISA, 97.78% results were same. The 466blood samples collected from Hubei province, Jiangsu province, New zealand, Australiaand Canada were examined for the presence of A. marginale by iELISA. The resultsshowed that the infection rate of A. marginale in Hubei province was 12.89%, nothingwas found in the sample of Jiangsu province, New zealand, Australia and Canada. Eperythrozoonosis is a zoonosis (anthropozoonosis) in many kinds of animals, whichattached to erythrocytes, dissociated in plasma and marrow. Formerly, E.wenyonii isclassified as the rickettsial agent. Phylogenetic analysis of 16S rRNA gene showed thateperythrozoon is closely related to mycoplasma recently. In resent years, the reportsassociated with this disease are increasing, it causes certain losses to animal husbandry.To date, E. wenyonii has not been cultivated. Laboratory confirmation of E. wenyoniistill relies on the microscopic examination of peripheral blood smears to directly visualizeE. wenyonii cells attached to erythrocytes. The drawbacks of microscopy includeproblems with both specificity and sensitivity, and it's very easy to present false positive.Serodiagnosis is hampered by the lack of standardized antigen preparation and the failureto discriminate present and past infection. Recently, molecular methods such as DNAhybridization and PCR assays have been developed to overcome problems associatedwith the low sensitivity in diagnosing chronic E. wenyonii with a low level of bacteraemiaby microscopy.We sought to develop a PCR to detect E. wenyonii in cattle. The specific 314bpfragment was amplified from 16S rRNA gene of E. wenyonii. The practical limit ofdetection showed that it had high sensitivity; an approximate DNA of 1.78 fg wasdetected by the PCR system and there were no cross among Eperythrozoon suis, Babesiaorientalis, Babesia bovis, B.bigemina, Anaplasma marginale, Toxoplasma gondii,Theileria sp. and E. wenyonii. The 309 blood samples collected from Hubei and Jiangsuprovince were examined for the presence of E. wenyonii by PCR. The results showed thataverage infection rate of E. wenyonii in Hubei and Jiangsu province was 10.3% and 3.5 %.This data revealed that bovine eperythrozoonsis present in Hubei and Jiangsu province.The results also showed that PCR was more sensitive than the examination ofmicroscope.